Many commercially useful microorganisms use glucose as their main carbohydrate source. The use of glucose by microorganisms developed for production of commercially desirable products does not generally provide a commercially desirable method for production due to the high cost of glucose. The use of fructose and mixed feedstocks containing fructose and other sugars as carbohydrate sources for microorganism production systems would be more commercially desirable, because these materials are more readily available at a lower cost. Desirable commercial feedstocks contain non-glucose breakdown products of starch or a variety of sugars such as fructose and xylose. Low cost feedstock derived from sucrose generally contains essentially equal amounts of glucose and fructose. Use of the fructose present in these types of feedstocks is desirable to obtain efficient production using a microorganism, such as in fermentation. However, microorganisms used to develop production microorganisms for making commercially desirable products do not generally have the ability to efficiently utilize fructose as a major carbohydrate source.
Though the most common sugar transport system found in prokaryotes, the phosphoenolpyruvate-dependent phosphotransferase system (PTS), is able to transport fructose, glucose is the preferred substrate. The PTS has been shown to mediate the efficient use of sugars through sensing and adjusting to sugar gradients and regulating expression of genes encoding enzymes responsible for uptake and metabolism of the various substrates (Stülke, J., and W. Hillen. 1999. Curr. Opin. Microbiol. 2:195-201; Mechanism and Regulation of Carbohydrate Transport in Bacteria, 1985, Academic Press, New York, M. H. Saiered., pp 70-74; Jacob, F. and J. Monod, 1961, J. Mol. Biol. 3:318-356). Bacterial cells show preferential sugar use, with glucose being most desirable. In artificial media containing every PTS sugar, glucose is metabolized to its entirety ahead of all other sugars (McGuinnis, J. F. and Palgen, K. 1969, J. Bacteriol. 100:902-913). Bacterial cells are adapted to recovering maximal energy from a substrate, and glucose provides slightly more energy compared to many other sugars, thus supporting a higher specific growth rate (Juana M. Gancedo, Microbiol Mol Biol Rev., 1998, 62:334-361).
In the PTS, sugars are phosphorylated during transport, and this activity is directly linked to the internal concentration of a glycolytic intermediate, phosphoenolpyruvate (PEP). PEP is the source for phosphate used to create a high-energy ester linkage that is necessary for subsequent sugar metabolism. The linkage of sugar transport and PEP limits the availability of PEP for use in synthesis of other products which are derived from PEP. For example, the yield of aromatic compounds produced by fermentation using E. coli is limited by the availability of PEP (Flores, N., Xiao, J., Berry, A., Bolivar, F. and F. Valle, 1996 Nat. Biotechnol. 14:620-623). Eliminating glucose uptake by PTS removes the connection between PEP and transport, making PEP available to biosynthetic pathways involved in the production of aromatics. Making use of an alternative (non-PTS) glucose transport system was demonstrated to be an effective method of increasing PEP levels and yield of compounds which use PEP as a precursor such as phenylalanine (Chen, R., Hatzimanikatis, V., Yap, W., Postma, P. and J. E. Bailey 1997, Biotechnol. Prog. 13:768-775).
Bypassing the PTS also conserves a molecule of ATP. Production strains of microorganisms have been developed which lack PTS and provide an alternative glucose assimilation system. In WO 2004/033471, a PTS−/Glu− (non glucose utilizing) bacterial host cell was converted to glucose utilizing capability by increasing the expression of an endogenous glucose assimilation protein. Specifically, a promoter with the ability to direct high expression was integrated into the genome adjacent to a galactose-proton symporter (galP) coding region. In addition, in this galP-engineered strain, a high expression promoter was integrated adjacent to a coding region for glucose kinase to provide enhanced glucose utilizing capability. In this report, it is suggested that other glucose phosphorylating enzymes may be overexpressed to provide enhanced glucose utilization. Fructokinases are mentioned as possibilities since these are inferred as having some glucose phosphorylating activity, but there is no mention of using fructose as a substrate.
In U.S. Published Patent Application No. 2001/0049126, strains of Escherichia that are not able to utilize sucrose were provided that capability by adding sucrose PTS genes or sucrose non-PTS genes. The introduction of these genes provided the recipient E. coli strain with the capability of growing on sucrose and producing the amino acid threonine. Different strains that had been engineered for production of various amino acids and that expressed the sucrose non-PTS genes were able to produce these amino acids when grown on sucrose.
In WO 98/18937, production of substances that use PEP as a precursor was improved by freeing PEP from use in phosphorylation during transport. PEP derivatives that are substances from aromatic metabolism are the products which this report addresses, specifically aromatic amino acids. Increased expression of glucokinase in a PTS+ strain of E. coli or of glucokinase and the Zymomonas mobilis glucose facilitator (glf) protein in a PTS− E. coli strain resulted in increased production of the amino acid phenylalanine with growth on glucose medium.
In Weisser et al. (1995, J. Bacteriol. 177:3351-3354), induced expression of fructokinase (frk) and glf in a PTS− E. coli strain did not support sustained growth of the cells on fructose. These cells that were engineered for high expression of the Zymomonas mobilis glf gene and the Zymomonas mobilis frk gene, doubled once and then stopped growing and eventually lysed. The authors speculate that a metabolic imbalance, such as accumulation of fructose-6-phosphate or draining of ATP, could be the reason for the inability to grow.
In U.S. Published Patent Application No. 2004/0152174A1, E. coli strains were engineered to produce high yields of 1,3-propanediol, using glucose as the carbon substrate. Suggestions of further suitable carbon substrates include lactose, sucrose, and fructose. However, fructokinase is not present in this disclosure.
Thus, the gene expression requirements for efficient fructose utilization in a PTS− microbial production host remain an unsolved issue. As described above, it is desirable to provide fructose utilization capability to PTS− microorganisms for growth on fructose during production of commercial products, because of the availability and lower cost of fructose-containing carbohydrate sources.